Nucleic acid association concerns techniques by which complementary base regions of two separate strands of nucleic acid, or distinct base regions of the same nucleic acid strand, anneal to each other through Watson-Crick base pairing, thus forming at least partially double-stranded nucleic acids. Examples of such pairing include two complementary DNA sequences, a single-stranded DNA sequence and a complementary RNA sequence, and two complementary RNA sequences, as well as modified forms of these nucleic acids, such as peptide nucleic acids or locked nucleic acids. See Nielsen et al, “Peptide Nucleic Acids,” U.S. Pat. No. 5,773,571; see also Imanishi et al., “Bicyclonucleoside and Oligonucleotide Analogues,” U.S. Pat. No. 6,268,490. For base pairing to take place, it is necessary to incubate the single-stranded nucleic acids under conditions which facilitate the formation of stable duplexes between nucleic acids or within a nucleic acid having complementary base regions. The formation of duplexes under stringent assay conditions can provide useful information about at least one member of each duplex, including the source from which that particular member was derived (e.g., a specific virus, microorganism, plant or animal), whether directly or by nucleic acid amplification, the presence of a disease-associated gene, the level of gene expression, the identification of genetic variability, including the detection of mutations and polymorphisms, and nucleic acid sequence information.
The rate at which nucleic acids having complementary base regions associate to form duplexes follows second-order kinetics. Thus, as the concentration of single-stranded nucleic acids is increased, the reaction rate (i.e., the rate at which duplexes are formed) is also increased. Conversely, as the concentration of single-stranded nucleic acids is decreased, the reaction rate is likewise decreased and, as a result, more time is required for the formation of double-stranded nucleic acids.
It is also well known that the temperature a reaction mixture affects the rate at which complementary nucleic acids associate. For example, as the temperature of a reaction mixture falls below the melting temperature or Tm (the temperature at which 50% of the duplexed molecules are rendered single-stranded), a maximum rate of reaction will be reached at temperatures approximately 15° to 30° C. below the Tm. Decreasing the temperature still further will lower the reaction rate below this maximum rate.
The reaction rate between nucleic acids having complementary base regions is also very dependent upon the ionic strength of a reaction mixture. Deoxynucleic acids, for instance, show a marked increase in reaction rates up to about 1.2 M NaCl, at which point the rate of association becomes essentially constant. See Wetmur et al. J. Mol. Biol. (1968) 31:349-379. And hybridizations between DNA and RNA molecules show a 5-6 fold increase in the rate of association when the ionic strength is increased from 0.2 to 1.5 M NaCl. The effect of changes in salt concentration on the rate of DNA:DNA reactions is greater than it is for RNA:DNA reactions.
A major limitation on the utility of many known nucleic acid association techniques is the basic rate of the reaction. Reaction times can be on the order of several hours to tens of hours, and even days in certain instances. Increasing the reaction rate by increasing the amount of single-stranded nucleic acid molecules in a reaction, in order to take advantage of second-order kinetics, is an undesirable solution for several reasons. First, in many applications the targeted nucleic acid in a reaction mixture has been extracted from a physiological sample, thereby imposing inherent limits on the amount of the targeted nucleic acid available in the reaction mixture, at least in the absence of an amplification procedure such as the polymerase chain reaction. See, e.g., Mullis, “Process for Amplifying Nucleic Acid Sequences,” U.S. Pat. No. 4,683,202. Second, there are considerable expenses associated with the use of nucleic acid reactants, and even greater expenses associated with a nucleic acid amplification procedure (e.g., enzymes and amplification primers), thus affecting the practicality of using more of these reactants or adding amplification reactants. Third, increasing the quantity of labeled, single-stranded nucleic acid probe molecules in a reaction mixture will cause a decrease in the sensitivity of a reaction, since the additional single-stranded nucleic acid probe molecules will elevate the background noise.
Accordingly, it is a principal object of the present invention to provide a method for forming a duplex from single-stranded regions of separate polynucleotides which enhances the association rate of the polynucleotides, whether reassociation or hybridization, and which enhancement can be demonstrated using reference conditions, including those conditions set forth herein. It is a further object of the present invention to provide a method for enhancing association rates that would be applicable to DNA:DNA, RNA:DNA and RNA:RNA reaction systems. Additionally, it is an object of the present invention to provide a method for promoting polynucleotide association rates that is applicable to reaction mixtures having a range of temperature and ionic strength conditions.